Micropropagation of black barhee date palm

ABSTRACT

Disclosed herein are media, kits, systems and methods for achieving micropropagation of a Black Barhee date palm on a commercially relevant scale. Compositions and methods for each stage of micropropagation, including initiation, elongation, and rooting are disclosed in the present application. Also disclosed are conditions for harvesting and sterilizing explant tissue for micropropagation.

FIELD OF THE INVENTION

The invention generally relates to media, kits, systems and methods forachieving micropropagation of Black Barhee date palms.

BACKGROUND

The following description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention.

The date tree is dioecious, i.e., female and male flowers develop ondifferent individuals. To produce fruits, the female trees have to bepollinated with pollen produced by the male tree. The fruits containviable seeds, but propagation by seed is time-consuming due toheterozygosity and resulting segregation. Conventional propagation iscarried out through vegetative cloning via so-called date palm offshoots(suckers). However, the number of offshoots produced by individual datepalms is limited and production restricted to early developmentalstages. The number of trees required to replace destructed, diseased ornewly planned orchards cannot be nearly produced through conventionalvegetative cloning. In addition, cloning through natural propagulesusually does not eliminate pathogens present in the parent plant butrather may lead to their accumulation. Only an appropriate in-vitrocloning (micropropagation) technique with strong multiplication resultshas the potential to deliver enough healthy trees for a timely relief ofthe shortage. In troubled areas, such a relief would significantlycontribute to the amelioration of hunger and desertification and wouldcontribute to society building and pacification. In areas with moreintact infrastructure, newly planted orchards can be of significanteconomic importance.

One variety of date which is considered especially important to the dateindustry is Black Barhee (also known as 8-Ball). Developed by BenLaflin, Jr., Black Barhee is the product of a pistillate Barhee crossedwith a fourth-generation-backcrossed staminate Medjool. The staminateMedjool was a product of the breeding program at the USDA Date andCitrus Station in Indio, Calif. Laflin obtained the staminate Medjoolwhen the station closed in 1980, and Black Barhee first fruited in the1990s. A very dark, nearly black date, Black Barhee is noted for itssoft, smooth flesh and pleasing flavor. The Black Barhee date palm isintermediate in appearance between the two parent varieties. BlackBarhee date palm lacks characteristic wings or ridges on the seed,belying its Medjool parentage. Although it produces significantly moreoffshoots than Benji, many offshoots of Black Barhee are high on thetrunk.

A non-limiting description of characteristics of Black Barhee date palmfruit is provided herein. One of skill in the art would realize that thedescription could vary somewhat for various reasons.

Fruit—Khalal red; rutab and tamar very dark brown, nearly black, withglaucous bloom lending a somewhat purplish cast; broadly ovate; 33-40mm×26-30 mm; skin medium, shrinking with and adhering to flesh inirregular folds; flesh moderately soft, smooth with little rag; flavorpleasing; ripening midseason.Seed—Brownish with some areas of gray; oblong-elliptic, broadest at orslightly distal of middle, apex rounded and prominently mucronate, basepointed; 16-24 mm×8-10 mm; germ pore central; furrow wide, shallow.

The difficulties encountered in the micropropagation of date palm,including Black Barhee, are the various physiological stages ofoffshoots coming from extreme climatic environments, the physiologicalshock produced by the rough harvest techniques for offshoots andflowers, the equally rough isolation technique for the apical explant,the high incidences of material contamination, the polyphenol productionand peroxidation of explants and problems related to hyperhydricity,dormancy, and positional bud effects (topophysis). There is a need inthe art for media, kits, systems and methods that overcome thesedifficulties, and allow for commercial-scale mass-cloning of date palms,including Black Barhee.

SUMMARY OF THE INVENTION

In various embodiments, the invention teaches a method formicropropagating a Black Barhee date palm. In some embodiments, themethod includes applying a first medium in vitro to a Black Barhee datepalm explant tissue that includes meristematic cells, at least until ashoot is initiated, wherein the first medium includes: (a)6-(gamma-gamma-dimethylallylamino)-purine (2-ip), 6-Benzylaminopurine(BAP) and Naphthoxyacetic acid (NOA); or (b) 2,4-Dichlorophenoxyaceticacid (2,4-D), 6-(gamma-gamma-dimethylallylamino)-purine (2-iP) andkinetic (KIN); or (c) 6-Benzylaminopurine (BAP), kinetic (KIN), and1-Naphthaleneacetic acid (NAA); and applying a second medium in vitro tothe initiated shoot, wherein the second medium includes6-Benzylaminopurine (BAP).

In some embodiments, the invention teaches a method for micropropagatinga Black Barhee date palm that includes applying a first medium in vitroto a Black Barhee date palm explant tissue that includes meristematiccells, until a shoot is initiated, wherein the first medium includes:(a) 6-(gamma-gamma-dimethylallylamino)-purine (2-ip),6-Benzylaminopurine (BAP) and Naphthoxyacetic acid (NOA); or (b)2,4-Dichlorophenoxyacetic acid (2,4-D),6-(gamma-gamma-dimethylallylamino)-purine (2-iP) and kinetic (KIN); or(c) 6-Benzylaminopurine (BAP), kinetic (KIN), and 1-Naphthaleneaceticacid (NAA). In some embodiments, the first and/or second media furtherincludes charcoal. In some embodiments, the first medium furtherincludes any of about 25%, about 50%, about 75%, about 90% or 100% ofMurashige and Skoog standard concentration of salts.

In various embodiments, the invention teaches a method formicropropagating a Black Barhee date palm that includes applying amedium in vitro to an initiated Black Barhee date palm shoot derivedfrom an explant, wherein the medium includes 6-Benzylaminopurine (BAP).In some embodiments, the medium further includes any of about 25%, about50%, about 75%, about 90% or 100% of Murashige and Skoog standardconcentration of salts. In certain embodiments, the Black Barhee datepalm is a male. In some embodiments, the Black Barhee date palm is afemale. In certain embodiments, the Black Barhee date palm explantincludes plant tissue from an offshoot of a Black Barhee date palm. Insome embodiments, the Black Barhee date palm explant includes planttissue from a flower, a leaf, or a bud of a Black Barhee date palm.

In various embodiments, the invention teaches a medium that includes theconstituents of PAE-1, PAE1-M, PAE-9, PAE-21, PAE-71, PAE-19 or PAE-7.

In various embodiments, the invention teaches a kit for micropropagatinga Black Barhee date palm. In some embodiments, the kit includes one ormore media that includes the constituents of a medium selected fromPAE-1, PAE-1M, PAE-71, PAE-9, PAE-19, PAE-21 and PAE-7. In someembodiments, the kit further includes a tissue of a Black Barhee datepalm. In some embodiments, the tissue includes plant tissue from anoffshoot, a flower, a leaf, or a bud of the Black Barhee date palm. Insome embodiments, the kit further includes instructions for the use ofthe one or more media for the purpose of micropropagating the BlackBarhee date palm.

In various embodiments, the invention teaches a Black Barhee date palmresulting from any of the methods described above.

In various embodiments, the invention teaches a Black Barhee date palmtissue resulting from any of the methods described above.

In various embodiments, the invention teaches a Black Barhee date palmcell resulting from any of the methods described above.

In various embodiments, the invention teaches a Black Barhee date palmplant resulting after exposing a tissue or a cell of a Black Barhee datepalm to media that includes the constituents of a medium selected fromPAE-1, PAE1-M, PAE-9, PAE-71, PAE-19, PAE-21 and PAE-7. In someembodiments, the origin of the tissue is an offshoot, a flower, a leaf,or a bud.

In various embodiments, the invention teaches a Black Barhee date palmtissue resulting after exposing a tissue or a cell of a Black Barheedate palm to media that includes the constituents of a medium selectedfrom PAE-1, PAE-1M, PAE-71, PAE-9, PAE-21, and PAE-7. In variousembodiments, the origin of the tissue of the Black Barhee date palm isan offshoot, a flower, a leaf, or a bud.

In various embodiments, the invention teaches a Black Barhee date palmplant cell resulting from exposing a tissue or a cell of a Black Barheedate palm to media that includes the constituents of a medium selectedfrom PAE-1, PAE-1M, PAE-71, PAE-9, PAE-21, PAE-19 and PAE-7. In certainembodiments, the tissue or the cell originates from a callus formed onan explant of a Black Barhee date palm. In some embodiments, the explantincludes tissue from a flower, a bud, a leaf, or an offshoot of a BlackBarhee date palm.

In various embodiments, the invention teaches a Black Barhee date palmplant resulting from exposing a globule of a callus of a Black Barheedate palm explant to a medium comprising the constituents of mediumselected from the group consisting of PAE-1, PAE-1M, PAE-9, PAE-19,PAE-21, and PAE-7, wherein a starting material from which the callus isformed is a flower, a bud, a leaf, or an offshoot of a Black Barhee datepalm.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments are illustrated in the referenced figures. It isintended that the embodiments and figures disclosed herein are to beconsidered illustrative rather than restrictive.

FIG. 1 depicts, in accordance with various embodiments of the invention,a schematic representation of the 4 general plant regeneration pathwaysof explants (a) direct organogenesis, (b) direct embryogenesis, (c)indirect organogenesis and (d) indirect embryogenesis. C=callus;E=explant.

FIG. 2 depicts, in accordance with various embodiments of the invention,an offshoot, weighing approximately 18 pounds (3 feet in length), withthe position where the shoot bud is situated in the core of the offshootmarked (white box). A cylindrical piece of approximately 6 cm by 8 cm,containing the shoot bud in its center, is excised, sterilized andfurther stripped under aseptic conditions until the bud (triangle) isuncovered. Offshoot removal and the initial steps of explant isolationrequire forceful manipulations. Great care must be taken not to injurethe core during these steps. Bar=10 mm.

FIG. 3 depicts, in accordance with various embodiments of the invention,tissue culture steps are performed in a laminar flow hood to createsterile working conditions. Prior to further excision, the isolatedoffshoot core is subjected to chemical sterilization. The cylindricalpiece is preferably large enough not to allow the damaging chemicals(bleach, alcohol) to penetrate the fragile bud. The picture illustratesthe further removal of leaves from the cylinder.

FIG. 4 depicts, in accordance with various embodiments of the invention,a bud (front, see also FIG. 2) and two adjacent leaf pieces on asolidified culture medium. Explants may be considerably smaller.

FIG. 5 depicts, in accordance with various embodiments of the invention,densely packed male (top) and female flowers (bottom) with protectivesheath (spathe) cut open (brown spathe visible at top). Flowers stalkstaken into tissue culture can be younger than those shown here.

FIG. 6 depicts, in accordance with various embodiments of the invention,regenerating explants showing growth and greening of secondary leaf buds(organogenesis).

FIG. 7 depicts, in accordance with various embodiments of the invention,regenerated date palm plant on rooting medium. The plant shown can betransferred to soil and carefully acclimatized.

FIG. 8 depicts, in accordance with various embodiments of the invention,embryogenic callus showing numerous globular structures (early embryos)that can be further multiplied and developed into mature embryos, eachone a potential date palm plant.

FIG. 9 depicts, in accordance with various embodiments of the invention,a bar graph indicating the number of explants of a Black Barhee datepalm successfully initiated (i.e. in which a plant organ or portionthereof is initiated (i.e. begins to grow) from the explant) on eachmedia type listed on the x-axis. A total of 10 explants were tested oneach medium. PAE1 is the same as PAE-1 described herein, PAE9 is thesame as PAE-9 described herein, and PAE7 is the same as PAE-7 describedherein.

FIG. 10 depicts, in accordance with various embodiments of theinvention, a bar graph indicating the multiplication factor (i.e. foldincrease in number of propagules produced per 8 weeks) for each mediatype on which Black Barhee date palm tissue was placed, as listed on thex-axis. A total of 10 explants were tested on each medium. PAE13 is thesame as PAE-13 described herein, PAE15 is the same as PAE-15 describedherein, PAE17 is the same as PAE-17 described herein, PAE19 is the sameas PAE-19 described herein, and PAE21 is the same as PAE-21 describedherein.

DESCRIPTION OF THE INVENTION

All references cited herein are incorporated by reference in theirentirety as though fully set forth. Unless defined otherwise, technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Smith, March's Advanced Organic Chemistry Reactions, Mechanismsand Structure 7^(th) ed., J. Wiley & Sons (2013); Jain et al., Date PalmBiotechnology, Springer (2011); Taiz et al., Plant Physiology andDevelopment 6th ed., Sinauer Associates, Inc. (2010), and Hodel, D. R.and D. V. Johnson. 2007. Imported and American Varieties of Dates(Phoenix dactlifera) in the United States. UC ANR Publication 3498.Oakland, Calif.: University of California will provide one skilled inthe art with a general guide to many of the terms used in the presentapplication.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. Indeed, the present invention is inno way limited to the methods and materials described.

As used herein, “2,4-D” means 2,4-Dichlorophenoxyacetic acid.

As used herein, “BAP” means 6-Benzylaminopurine.

As used herein, “NAA” means 1-Naphthaleneacetic acid.

As used herein, “2iP” means 6-(gamma,gamma-Dimethylallylamino)purine.

As used herein, “NOA” means Naphthoxyacetic acid.

As used herein, the term “date palm” refers to Black Barhee date palm.Both male and female Black Barhee date palms are intended to be includedwithin the meaning of the term “date palm.”

As indicated above, embodiments disclosed herein provide for themicropropagation or tissue culturing (these terms are usedinterchangeably herein) of Black Barhee date palms on a commercialscale. Each discrete step described below and associated withmicropropagation may be utilized in conjunction with protocols otherthan those described herein. Thus, for example, the inventive harvestingor sterilization techniques described herein may be implemented inconjunction with methods and media different than those specifically setforth or referenced herein, and therefore represent “stand alone”contributions to the field.

Harvesting & Tissue Preparation Physiological Quality of Donor-Plants

Preferably, offshoots are taken from a professionally managed orchardwith no known infestation of Black Barhee date palm.

Explant Types

By way of non-limiting examples, explants can be isolated fromoffshoots, male and female flowers, or flowers from offshoots.

Physiological Quality of the Explants

Preferably, explants should be white in nature with little or noyellowish color.

Harvesting Procedure

In some embodiments, before flowers are open the inflourescence aretaken with outer layers removed to expose the flowers. Flowers are thendisinfected according to the protocols described herein or bytraditional protocols.

Stress-Relieving Treatment

The process of offshoot/flower harvesting, including the transport tothe culture facility, is extremely stressful to plant tissue.Physiological changes can be expected. Although it is known in planttissue culture, that so-called “unspecific shocks” (short heat-, cold-,salt-, dark-, etc.-treatments) can have a positive effect on tissue/cellregeneration, it is evident through experimental results that the“harvesting shock” should be minimized. In some embodiments, a “restingperiod” of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days between harvest ofthe offshoots/flowers and explant extraction may be implemented. In someembodiments, the resting period is implemented in the dark under mildand clean environmental conditions.

Black Barhee date palm Micropropagation Utilizing Offshoot StartingMaterial

Offshoot Extraction

Outer layers of offshoots from a Black Barhee date palm tree are removedgradually, and fibrous tissues are removed until the shoot tip area isexposed. The shoot tip is then excised by cutting a circle around thebase of the cylindrical shoot tip at an angle of approximately 45degrees.

Offshoot Sterilization

In various embodiments, carefully extracted apex-containing core piecesof offshoots (“offshoot samples”) are treated for 1-2 or more hours withantioxidant solution. In some embodiments, the offshoot samples aretreated for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more hours. In someembodiments, the antioxidant solution includes 50-200 mg/L of ascorbicacid and/or citric acid. In certain embodiments, the antioxidantsolution includes 150 mg each per liter of ascorbic acid and citricacid.

In certain embodiments, after treatment with antioxidant solution, theoffshoot samples are gas sterilized for 5-240, 10-120, 20-60 or 30-40minutes. In some embodiments, the offshoot samples are then gassterilized for 45-120 minutes. In certain embodiments, the duration ofgas sterilization is determined based on the offshoot condition (e.g.,tissue softness and color etc.). Merely by way of example, in terms oftissue softness, if the tissue is soft, then treatment is for 30-45minutes. On the other hand, if the tissue is hard then treatment is for20-60 minutes. In some embodiments, gas sterilization is carried out ina closed container in which chlorine gas is released through thereaction of bleach with concentrated hydrochloric acid. In someembodiments, the container used for gas sterilization is made of amaterial that may include, but is no way limited to plastic, glass,rubber, composite, and the like. The purpose of the gas sterilizationmethod is that it provides a mild way of penetrating small cavities ofthe tissue that are not reachable by liquid sterilization due to surfacetension.

In some embodiments, after gas sterilization, the offshoot sample ismoved into a laminar-flow hood for a 2-40, 4-30 or 6-20 min treatmentwith sodium hypochloride. In some embodiments, the sodium hypochloridetreatment is applied for 12-15 min. In certain embodiments, 0.01%-1.0%,0.08%-0.8%, 0.1%-0.6%, 0.4%-0.8%, 0.4%-0.6%, 0.5%-0.6% or 0.6%-0.7%sodium hypochloride is applied. In some embodiments, about 0.6% sodiumhypochloride is applied for 12-15 minutes.

In some embodiments, the offshoot sample is then washed with 1-6, 2-5 or3-4 rinses with sterilized reverse osmosis (RO) water. In certainembodiments, after 3 rinses with sterilized RO water, the sterilizationprocedure is complete.

In certain embodiments, the aforementioned offshoot sterilizationprotocols are applied to an offshoot of a Black Barhee date palm. In yetadditional embodiments, the above-described sterilization methods arebelieved to be useful for the sterilization of offshoots of other planttypes.

Offshoot Culture

In some embodiments, the sterilized core (sterilized as described aboveor otherwise) is carefully peeled by removal of the leaves until a sizeof approximately 1.2 cm in diameter and approximately 0.5 cm to 1.5 cmin length (cylinder-like) is reached. In certain embodiments, thecylinder is then cut into pieces longitudinally. In some embodiments,the cylinder is cut into 2-10, 4-9 or 6-8 pieces. In some embodiments,the method produces 1 piece that contain most of the primary apex andadditional pieces with secondary apices which may be leaf or flowerprimordia.

In certain embodiments, the explants are then cultured in groups of 1-6,2-5 or 3-4, in 10 cm Petri plates (or plates of another convenient size)in the dark at 23-29° C., 24-28° C. or 25-27° C. on an agar-solidifiedinitiation culture medium (e.g. a medium described in Table 4 or Tables7-11), and subcultured to the same or a different initiation culturemedium every 1-8, 3-7 or 4-6 weeks. In some embodiments, the explantsare subcultured every 4 weeks. In some embodiments, the initiationmedium for one or more subcultures is a medium described in Table 4, orany medium of Tables 7-11. In some embodiments, the initiation mediumdoes not contain 2,4-D. In certain embodiments, the initiation mediumcontains one or more organics that may include, but are in no waylimited to glutamine, adenine, and calcium pantothenate. In certainnon-limiting embodiments, the explant is initially cultured in a mediumthat includes M7, 1/2M7, M20, 1/2M20, M26, M27, M29, 1/2M52, PA01, PA02,PA03, PA04, PA05, PA06, PA07, PAE-1, PAE-1M (this medium is identical toPAE-1, except the concentration of NOA is reduced to 3 mg/L), PAE-9,PAE-71 (this medium is identical to PAE-7, except NAA is omitted and NOAis added at a concentration of 3 mg/L), or PAE-7. In an embodiment, theexplant is initially cultured in a medium that includes PAE-1. In anembodiment, the explant is initially cultured in a medium that includesPAE-1M. In an embodiment, the explant is initially cultured in a mediumthat includes PAE-7. In an embodiment, the explant is initially culturedin a medium that includes PAE-71. In an embodiment, the explant isinitially cultured in a medium that includes PAE-9. In some embodiments,the explant is subcultured during the initiation stage in a medium thatincludes M7, 1/2M7, M20, 1/2M20, M26, M27, M29, 1/2M52, PA01, PA02,PA03, PA04, PA05, PA06, PA07, PAE-1, PAE-1M, PAE-9, PAE-71, or PAE-7.Thus, one or more media may be utilized during the initiation stage. Inan embodiment, the explant is subcultured in a medium that includesPAE-1. In an embodiment, the explant is subcultured in a medium thatincludes PAE-1M. In an embodiment, the explant is subcultured in amedium that includes PAE-7. In an embodiment, the explant is subculturedin a medium that includes PAE-71. In an embodiment, the explant issubcultured in a medium that includes PAE-9. In some embodiments, theexplant is initially cultured or subcultured in a medium in which one ormore constituents of the above-mentioned media is modified by +0.5-20%(w/v), +2-15% (w/v), or +5-10% (w/v). In some embodiments, the media inwhich the explant is cultured or subcultured includes charcoal. In someembodiments, explants are subcultured every 1-8 weeks, 3-7 weeks, 4-6weeks, 4-7 weeks, 5-6 weeks, 6-7 weeks or 5-7 weeks, or more or lessfrequently, depending upon the condition of the explant. In someembodiments, the above-described initiation protocols are utilized inthe furtherance of organogenesis. In some embodiments, theabove-described initiation protocols are utilized in the furtherance ofembryogenesis. In some embodiments, if organogenesis is desired, thenthe explants are exposed to light after a period of 4-10 months(preferably 4-6 months). In some embodiments, if embryogenesis isdesired, then one or more individual globules are subcultured onto freshmedia prior to exposure to light at the end of the 4-10 month period(preferably 4-6 months).

Shoot-Multiplication

In some embodiments, freshly cultured offshoots treated according to theinventive method have a dormancy of 4-10 months during which theexplants increase through swelling (water uptake) but rarely display anyform of growth. Occasionally, some callus is produced from woundedtissue. In certain embodiments, the cultures are then transferred tolight. In some embodiments, the light conditions can be 2000-3000 lux.In some embodiments, light exposure induces organogenesis. In someembodiments, successful initiation is the formation of an organ, orportions thereof (i.e. a shoot or portion thereof), which can bedescribed as initiated material. In some embodiments, initiated materialis segregated from the starting explant material and optionally culturedand subcultured during the multiplication stage. In some embodiments,initiation may occur without light stimulation. In some embodiments, thetemperature applied at this stage is approximately 25-28° C. In someembodiments, the initial culturing and/or subculturing during themultiplication stage occurs on a medium listed in Table 5 or Tables7-13. In some embodiments, the initial culturing and/or subculturingduring the multiplication stage occurs on a medium listed in Table 12.In some embodiments, the initial culturing and/or subculturing duringthe multiplication stage occurs on a medium listed in Table 13. In someembodiments, the medium used during the multiplication stage is PAE-21.In some embodiments, the medium used during the multiplication stage isPAE-19. In some embodiments, during the multiplication stage theinitiated plant tissue is initially cultured or subcultured in a mediumin which one or more constituents of the above-mentioned multiplicationmedia is modified by ±0.5-20% (w/v), ±2-15% (w/v), or ±5-10% (w/v). Insome embodiments, the media in which the initiated tissue is cultured orsubcultured includes charcoal. In some embodiments, initial culturingand/or subculturing during the multiplication stage occurs on a mediumof Table 5 or Tables 7-11 which has been modified by reducing theconcentration of one or more hormones to ¾-⅛ of the initialconcentration, or ½-⅛ of the initial concentration. In some embodiments,the concentrations of hormones in the media listed in Table 5 or Tables7-11 are reduced to ⅕ of the initial concentration during themultiplication stage. In some embodiments, initial culturing and/orsubculturing during the multiplication stage occurs on a medium of Table5 or Tables 7-11 which has been modified by reducing the concentrationof every constituent except the organics to ¾-⅛ of the initialconcentration, or ½-⅛ of the initial concentration. In some embodiments,the concentrations of all constituents except for the organics in themedia listed in Table 5 or Tables 7-11 are reduced to ⅕ of the initialconcentration during the multiplication stage. In some embodiments, thesame medium is utilized during initial culturing and subsequentsubculturing. In other embodiments, a different medium is utilized forinitial culturing and subsequent subculturing.

In some embodiments, by applying the offshoot culture techniquesdescribed herein, after about 2, 3, 4, 5 or 6 months, a single culture(1 culture vessel derived from 1 explant piece) will have produced 10,20, 30, 40, 50, 60, 70, 80, 90, 100 or more subcultures (˜10, 20, 30,40, 50, 60, 70, 80, 90, 100 or more containers). In some embodiments,production lines are subcultured every 1, 2, 3, 4, 5, 6 or more weeksduring the multiplication phase. In some embodiments, production linesare subcultured every 4 weeks. Individual plantlets produced by theproduction line (usually approximately 6-10 cm in height and greaterthan about 0.5 cm in stem diameter) are carefully removed and treatedfor rooting.

Rooting

In certain embodiments, plantlets, approximately 3-15 cm, 5-12 cm or6-10 cm in height, and approximately 0.2-0.8 cm, 0.3-0.6 cm or 0.4-0.5cm in stem diameter, are carefully separated from the multiplicationcultures for rooting. In some embodiments, the preferred stem diameteris approximately 0.5 cm. Importantly, the rooting techniques describedherein often result in almost 100% rooting, which is unusually high.

In some embodiments, the club-shaped terminal of the shoot is identifiedand carefully cleaned from adhering leaflets and tissue. In certainembodiments, a clean cut is then applied to expose the meristem withoutdamaging it and it is inserted into rooting medium. In some embodiments,the rooting medium includes a rooting medium described in Table 6, or inthe examples set forth herein. In some embodiments, the rooting mediumis M70 or M82. In some embodiments, the rooting medium includes NAA at aconcentration of 0.1 mg/L and sucrose at a concentration of 40-60 g/L.In some embodiments, the concentration of NAA and/or sucrose aremodified by approximately up to +20%.

Black Barhee Date Palm Micropropagation Utilizing Flower StartingMaterial Flower Sterilization

In certain embodiments, carefully extracted fully closed flower spathesare washed with water and transferred to a laminar-flow hood. Inpreferred embodiments, the flower spathes are washed in hand-warm waterand transferred to the laminar-flow hood. In some embodiments, once theflowers spathes are transferred to the laminar-flow hood, they arewrapped in a bleach-saturated absorbent material and enclosed in anadditional layer of material to ensure proper contact with the bleach.In some embodiments, the flower spathes are wrapped in bleach-saturatedpaper towels and enclosed in aluminum foil to ensure proper contact withthe bleach. In some embodiments, the composition of the commercialbleach is approximately 4-6% NaClO by weight. In some embodiments, theduration of bleach treatment ranges from 1-30 minutes, 5-20 minutes or10-15 minutes. In some embodiments, the duration of treatment is 5 to 15min, depending on the size of the spathe. Merely by way of example, ifthe spathe is approximately 10 cm then it may be treated forapproximately 5 minutes, whereas if the spathe is approximately 40 cmthen it may be treated for approximately 15 minutes or more.

In certain embodiments, after bleach treatment, the spathe is carefullyopened with a scalpel and the flower strings are removed with forceps.In some embodiments, including if the flower strings are too long, theyare cut into approximately 5 to 10 cm pieces and treated withantioxidant solution (as described above in the protocol for offshoots)for 5-120 minutes, 10-90 minutes, 20-80 minutes or 30-60 minutes. Incertain embodiments, once the flower strings are treated withantioxidant solution, they are rinsed with sterilized water to completethe sterilization procedure. Importantly, the sterilization proceduresdescribed above can be implemented for male and female flowers alike.

In certain embodiments, the aforementioned flower sterilizationprotocols are applied to a Black Barhee date palm. In yet additionalembodiments, the above-described flower sterilization methods can beimplemented for the sterilization of flowers of other plant types.

Flower Culture

In certain embodiments, the sterilized flower strings (regardless ofsex) are cut into 0.5-2.5 cm, 0.75-1.75 cm or 1.0-1.5 cm segments, andcultured in groups of 5-30, 10-25 or 15-20 segments in 10 cm Petriplates in the dark at 23-29° C., 24-27° C. or 25-26° C. onagar-solidified initiation culture medium, and subcultured to the sameor a different medium every 2-8, 3-7 or 4-6 weeks. In some embodiments,the initiation medium for the flower culture includes one or more medialisted in Table 4 or Tables 7-11. In some embodiments, the flowerculture includes PAE-9. In some embodiments, the flower culture includesPAE-1M. In some embodiments, the subculturing occurs on the same mediumon which the flower string segment was initially placed. In certainembodiments the subculturing occurs on a different medium which includesone or more media listed in Table 4 or Tables 7-11. In some embodiments,the flower subculturing is on a medium that includes PAE-9. In someembodiments, the flower subculturing is on a medium that includesPAE-1M. In some embodiments, the flower subculturing is on a medium thatincludes PAE-1. In some embodiments, the flower tissue is initiallycultured or subcultured in a medium in which one or more constituents ofthe above-mentioned initiation media is modified by ±0.5-20% (w/v),±2-15% (w/v), or +5-10% (w/v). In some embodiments, the initialinitiation medium and/or subculturing medium includes charcoal.

Culture of In Vitro Flowers

When the inventive methods and media described above are implemented,plantlets regenerated in vitro from offshoots can spontaneously produceflowers in vitro. These flowers do not develop in a spathe but ratherform a naked single flower or a short naked string of flowers. Theseflowers, without sterilization or other treatments can be directlycultured again as described in the section above describing flowerculture. In certain embodiments, when treated with the inventive mediaand methods, these cultures react relatively quickly and show amuch-reduced dormancy compared to normal.

Flower Multiplication

In various embodiments, flowers freshly cultured utilizing the methodsand media described herein have of dormancy of approximately 4-10months, during which the explants increase through swelling (wateruptake) but rarely display any form of growth. Occasionally, when theinventive methods are implemented, some callus is produced from woundedtissue. After the dormancy phase is completed, the explant producesvigorous callus or direct embryos or callus-derived embryos depending onthe medium. In certain embodiments, the receptacle is the regeneratingflower part. By implementing certain embodiments of the inventivemethod, about 2 flowers per plate regenerate (from about 50 female orabout 150 male flowers per plate). In certain embodiments, the culturesare then transferred to light. Merely by way of example, the lightconditions can be 2000-3000 lux. In some embodiments, light is providedin 10-14 hour cycles during the multiplication phase. In someembodiments, light is provided in 12 hour cycles during themultiplication phase. In some embodiments, the temperature for this stepis approximately 25-28° C. In some embodiments, by implementing theinventive methods described above, in approximately 2, 3, 4, 5, 6 monthsor more a single culture (1 culture vessel derived from 1 explant piece)will have produced 10, 20, 30, 40 or more subcultures (10, 20, 30, 40 ormore containers). These cultures usually contain a mixture of embryosand shoots. In some embodiments, if the embryo rate is high, subcultureproduction is increased proportionally. In some embodiments, productionlines are subcultured every 2-6, 3-5 or 4 weeks during thismultiplication stage. Individual plantlets produced by the productionline (which are usually about 6-10 cm in height and more than 0.5 cm instem diameter) are carefully removed and treated for rooting. In someembodiments, initial culturing and/or subculturing during themultiplication stage occurs on a medium of Table 5 or Tables 7-11 whichhas been modified by reducing the concentration of one or more hormonesto ¾-⅛ of the initial concentration, or ½-⅛ of the initialconcentration. In some embodiments, the concentrations of one or morehormones in the media listed in Table 5 or Tables 7-11 are reduced to ⅕of the initial concentration during the multiplication stage. In someembodiments, initial culturing and/or subculturing during themultiplication stage occurs on a medium of Table 5 or Tables 7-11 whichhas been modified by reducing the concentration of all but the organicconstituents to ¾-⅛ of the initial concentration, or ½-⅛ of the initialconcentration. In some embodiments, the concentrations of allconstituents but the organics in the media listed in Table 5 or Tables7-11 are reduced to ⅕ of the initial concentration during themultiplication stage. In some embodiments, the same medium is utilizedduring initial culturing and subsequent subculturing. In otherembodiments, a different medium is utilized for initial culturing andsubsequent subculturing.

Rooting

In some embodiments, plantlets, 3-15 cm, 5-12 cm or 6-10 cm in heightand approximately 0.5 cm in stem diameter are carefully separated fromthe multiplication cultures described above in this section for rooting.The rooting techniques described herein result in almost 100% rooting,which is unusually high for Black Barhee date palms. In someembodiments, the club-shaped terminal of the shoot is identified andcarefully cleaned from adhering leaflets and tissue. In certainembodiments, a clean cut is then applied to expose the meristem withoutdamaging it and it is inserted into rooting medium. In some embodiments,the rooting medium includes a rooting medium described in Table 6, or inthe examples set forth herein. In some embodiments, the rooting mediumis M70 or M82. In some embodiments, the rooting medium includes NAA at aconcentration of 0.1 mg/L and sucrose at a concentration of 40-60 g/L.In some embodiments, the concentration of NAA and/or sucrose aremodified by approximately up to +20%.

Indoor Acclimation

In some embodiments, the plantlets are allowed to grow for approximately3 months under same 10-14 or 12 hour cycle light conditions as above,until approximately 6 inch plants are developed with a substantialnumber of roots.

Outdoor Acclimation and Establishment

In some embodiments, plants are then transfer to the greenhouse pottedinto soil. In some embodiments, plants are then planted into the groundafter approximately 6-8 months depending on the progress of theirgrowth.

Culture Media

Micropropagated plants are generally grown in vitro in sterile media.The sterile media can be liquid, semi-solid, or solid, and the physicalstate of the media can be varied by the incorporation of one or moregelling agents. Any gelling agent known in the art that is suitable foruse in plant tissue culture media can be used in conjunction with theinventive media. In some embodiments, agar is used for this purpose.Examples of such agars include Agar Type A, E or M and Bacto® Agar.Other exemplary gelling agents include carrageenan, gellan gum(commercially available as PhytaGel™, Gelrite® and GelZan™), alginicacid and its salts, and agarose. Blends of these agents, such as two ormore of agar, carrageenan, gellan gum, agarose and alginic acid or asalt thereof also can be used. In certain non-limiting examples, theagar utilized is made by Sigma™ Typically, the media used in conjunctionwith the inventive methods comprises agar with the addition of variouscompounds such as nutrients, inorganic salts, growth regulators, sugars,vitamins and other compounds. Other media additives can include, but arenot limited to, amino acids, macroelements, iron, microelements,inositol and undefined media components such as casein hydrolysates oryeast extracts. For example, the media can include any combination ofNH₄NO₃; KNO₃; Ca(NO₃)₂; K₂SO₄; MgSO₄; MnSO₄; ZnSO₄; CuSO₄; CaCl₂; KI;CoC1 ₂; H₃BO₃; Na₂MoO₄; KH₂PO₄; FeSO₄; Na₂EDTA; Na₂H₂PO₄; myoinositol;thiamine; pyridoxine; nicotinic acid; glycine; glutamine, adenine,calcium pantothenate, riboflavin; ascorbic acid; silicon standardsolution; 3-naphthoxyacetic acid (NAA); indole butyric acid (IBA); 3-indoleacetic acid (IAA); benzylaminopurine (BAP);6-(gamma-gamma-dimethylallylamino)-purine (2-ip); sugar; agar;carrageenan and charcoal. Non-limiting examples of plant growthregulators include auxins and compounds with auxin-like activity,cytokinins and compounds with cytokinin-like activity. Exemplary auxinsinclude 2,4-dichlorophenoxyacetic acid, IBA, picloram and combinationsthereof. Exemplary cytokinins, in addition to meta-topolin andthidiazuron, include adenine hemisulfate, benzyladenine,dimethylallyladenine, kinetin, zeatin and combinations thereof.Gibberellic acid also can be included in the media. One or more sugarcan be included in the media and can serve as a carbon source.Non-limiting examples of sugars that may be used include sucrose,glucose, maltose, galactose and sorbitol or combinations thereof.

In various embodiments, including those set forth in Tables 4-9, mediadescribed herein are Murashige and Skoog (MS) based. Tables 1-3 indicatethe major salts, minor salts, and vitamins and organics of Murashige andSkoog Media, with the exception of sucrose, which is optionally includedhere in the referenced concentration range of Table 3 or in the othermedia described in Tables 4-13. The category of organics in MS media mayfurther include indole acetic acid (1-30 mg/l), kinetin (0.04-10 mg/l)and lactalbumin hydrolysate (edamin). For each medium described inTables 4-9, major salts (macronutrients), minor salts (micronutrients),and organics are included. Concentrations of major salts included ineach medium of Tables 4-9 are listed in Table 1, unless medium-specificmodifications are indicated (See for example Tables 4-6 and 10-13).Concentrations of minor salts included in each medium of Tables 4-9 arelisted in Table 2, unless medium-specific modifications are indicated(See for example Tables 4-6 and 10-13). Concentrations of organicsincluded in each medium of Tables 4-6 are listed in Table 3, unlessmedium-specific modifications are indicated (See Tables 4-6). In eachmedium of Tables 4-6, agar is added to a concentration of 6.4±2 g/L,unless medium specific modifications are indicated (See Tables 4-6). Insome embodiments, the pH of the media listed in Tables 4-9 or otherwisereferenced herein is a pH that is generally hospitable to plants(typically from 4.0-7.0 or 4.5-6.5). In certain embodiments, the pH ofthe media described herein is adjusted to 5.5-6.3, 5.6-6.2, 5.7-6.1 or5.8-6.0. In some embodiments, the pH of the media is adjusted to 5.7.

Although relatively narrow concentrations of the cytokinins and auxinsare included in the media set forth in Tables 4-13, all of thecytokinins and auxins listed may be present in a range ofconcentrations.

Merely by way of example NAA, NOA, BAP, 2,4-D, IBA, IAA and 2iP can bepresent at 0.001 mg/L, 0.01, 0.1, 1, 2, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1,5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or100 mg/L or 0.001 mg/L, 0.01, 0.025, 0.05, 0.075, 0.1, 0.15, 0.2, 0.25,0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9,0.95, 1.0, 1.25, 1.50, 1.75, 2.25, 2.5, 2.75, 3.5, 4.5, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99or 100 mg/L.

A pharmaceutically acceptable salt is any salt of the parent compoundthat is suitable for use in the methods disclosed herein. Apharmaceutically acceptable salt also refers to any salt which may formas a result of administration of an acid, another salt, or a prodrugwhich is converted into an acid or salt. A salt comprises one or moreionic forms of the compound, such as a conjugate acid or base,associated with one or more corresponding counter-ions. Salts can formfrom or incorporate one or more deprotonated acidic groups (egcarboxylic acids), one or more protonated basic groups (eg amines), orboth (e.g. Zwitterions). Not intended to be limited by the abovedescribed compounds, various tautomers of the above compounds may bepossible. As used herein, “tautomer” refers to the migration of protonsbetween adjacent single and double bonds. The tautomerization process isreversible. Other tautomers are possible when the compound includes, forexample but not limited to, enol, keto, lactamin, amide, imidic acid,amine, and imine groups. Tautomers will generally reach an equilibriumstate wherein the double bond is resonantly shared between the two bondlengths.

With the foregoing descriptions in mind, in various embodiments theinvention teaches a medium for use in micropropagating a Black Barheedate palm, wherein the medium comprises, consists of, or consistsessentially the constituents of any medium of Tables 4-13. In someembodiments, the concentrations of one or more of any of theconstituents of Tables 4-13 may be modified by up to approximately +20%of the concentration specifically listed. In some embodiments, themedium is used to micropropagate a Black Barhee date palm. In someembodiments, the medium for use in micropropagating a Black Barhee datepalm comprises, consists of, or consists essentially of the constituentsof PAE-1, PAE-1M, PAE-9, PAE-71, PAE-21, PAE-19 or PAE-7.

In various embodiments, the invention teaches a method formicropropagating a Black Barhee date palm. In some embodiments, themethod includes applying a first medium in vitro to a Black Barhee datepalm explant tissue that includes meristematic cells, until a shoot isinitiated, wherein the first medium includes: (a)6-(gamma-gamma-dimethylallylamino)-purine (2-iP), 6-Benzylaminopurine(BAP) and Naphthoxyacetic acid (NOA); or (b) 2,4-Dichlorophenoxyaceticacid (2,4-D), 6-(gamma-gamma-dimethylallylamino)-purine (2-iP) andkinetic (KIN); or (c) 6-Benzylaminopurine (BAP), kinetic (KIN),1-Naphthaleneacetic acid (NAA). In some embodiments, the method furtherincludes applying a second medium in vitro to the initiated shoot,wherein the second medium includes 6-Benzylaminopurine (BAP). In certainembodiments, the first and/or second media further includes charcoal. Insome embodiments, the first medium includes6-(gamma-gamma-dimethylallylamino)-purine (2-iP), 6-Benzylaminopurine(BAP) and Naphthoxyacetic acid (NOA). In some embodiments, the firstmedium includes 2-iP at a concentration of 1-3 mg/L, BAP at aconcentration of 0.5-1.5 mg/L, and NOA at a concentration of 2.0-6.5mg/L. In an embodiment, the first medium includes 2-iP at aconcentration of 2 mg/L, BAP at a concentration of 1 mg/L, and NOA at aconcentration of 5 mg/L. In another embodiment, the first mediumincludes 2-iP at a concentration of 2 mg/L, BAP at a concentration of 1mg/L, and NOA at a concentration of 3 mg/L. In other embodiments, thefirst medium includes 2,4-Dichlorophenoxyacetic acid (2,4-D),6-(gamma-gamma-dimethylallylamino)-purine (2-iP) and kinetic (KIN). Insome embodiments, the first medium includes 2,4-D at a concentration of0.5-1.5 mg/L, 2-iP at a concentration of 2-4 mg/L, and KIN at aconcentration of 2-4 mg/L. In an embodiment, the first medium includes2,4-D at a concentration of 1 mg/L, 2-iP at a concentration of 3 mg/L,and KIN at a concentration of 3 mg/L. In yet other embodiments, thefirst medium includes 6-Benzylaminopurine (BAP), kinetic (KIN), and1-Naphthaleneacetic acid (NAA). In some embodiments, the first mediumincludes 6-Benzylaminopurine (BAP) at a concentration of 0.05-0.15 mg/L,kinetic (KIN) at a concentration of 0.005-0.015 mg/L, and1-Naphthaleneacetic acid (NAA) at a concentration of 0.5-1.5 mg/L. In anembodiment, the first medium includes 6-Benzylaminopurine (BAP) at aconcentration of 0.1 mg/L, kinetic (KIN) at a concentration of 0.1 mg/L,and 1-Naphthaleneacetic acid (NAA) at a concentration of 1 mg/L. Inadditional embodiments, the first medium includes BAP at a concentrationof 0.05-0.15 mg/L, KIN at a concentration of 0.005-0.015 mg/L, and2-Naphthoxyacetic acid (NOA) at a concentration of 2-4 mg/L. In anembodiment, the first medium includes BAP at a concentration of 0.1mg/L, KIN at a concentration of 0.01 mg/L, and 2-Naphthoxyacetic acid(NOA) at a concentration of 3 mg/L. In certain embodiments, the firstmedium further includes any of about 25%, about 50%, about 75%, about90% or 100% of Murashige and Skoog standard concentrations of majorsalts and/or minor salts and/or organics. In some embodiments, theconcentration of Murashige and Skoog standard concentrations of majorsalts and/or minor salts and/or organics is 20-100%, 30-90%, 40-80%, or50-70%. In certain embodiments, the concentrations of major salts and/orminor salts and/or organics in the first medium are according to theconcentrations of those constituents listed in Table 10 for PAE-1,PAE-9, or PAE-7. In some embodiments the concentrations of major saltsand/or minor salts and/or organics of the first medium are within +20%of the concentrations of those constituents listed in Table 10 forPAE-1, PAE-9, or PAE-7. In certain embodiments, the first mediumcomprises, consists of, or consists essentially of the components ofPAE-1, PAE-1M, PAE-9, PAE-7, or PAE-71. In some embodiments, the secondmedium includes BAP at a concentration of 0.5-1.5 mg/L. In someembodiments, the second medium includes BAP at a concentration of 1mg/L. In certain embodiments, the second medium further includes any ofabout 25%, about 50%, about 75%, about 90% or 100% of Murashige andSkoog standard concentrations of major salts and/or minor salts and/ororganics. In some embodiments, the concentration of Murashige and Skoogstandard concentrations of major salts and/or minor salts and/ororganics is 20-100%, 30-90%, 40-80%, or 50-70%. In certain embodiments,the concentrations of major salts and/or minor salts and/or organics inthe second medium are according to the concentrations of thoseconstituents listed in Table 12 for PAE-19 or PAE-21. In someembodiments the concentrations of major salts and/or minor salts and/ororganics of the second medium are within +20% of the concentrations ofthose constituents listed in Table 12 for PAE-19 or PAE-21. In certainembodiments, the second medium comprises, consists of, or consistsessentially of the components of PAE-19 or PAE-21 listed in Table 12. Insome embodiments, the second medium comprises, consists of, or consistsessentially of the components of PAE-21. In some embodiments, the secondmedium comprises, consists of, or consists essentially of PAE-19. Insome embodiments, after initiation and multiplication are accomplished,the tissue is subject to rooting (and optionally elongation prior torooting). In some embodiments, rooting is accomplished byexposing/incubating the multiplied tissue to any rooting mediumdescribed herein.

In various embodiments, the invention teaches a method formicropropagating a Black Barhee date palm. In some embodiments, themethod includes applying a medium in vitro to an initiated Black Barheedate palm shoot derived from an explant, wherein the medium includes6-Benzylaminopurine (BAP). In some embodiments, the medium includes BAPat a concentration of 0.5-1.5 mg/L. In some embodiments, the mediumincludes BAP at a concentration of 1 mg/L. In certain embodiments, themedium further includes any of about 25%, about 50%, about 75%, about90% or 100% of Murashige and Skoog standard concentrations of majorsalts and/or minor salts and/or organics. In some embodiments, theconcentration of Murashige and Skoog standard concentrations of majorsalts and/or minor salts and/or organics is 20-100%, 30-90%, 40-80%, or50-70%. In certain embodiments, the concentrations of major salts and/orminor salts and/or organics in the medium are according to theconcentrations of those constituents listed in Table 12 for PAE-19 orPAE-21. In some embodiments the concentrations of major salts and/orminor salts and/or organics of the medium are within +20% of theconcentrations of those constituents listed in Table 12 for PAE-19 orPAE-21. In certain embodiments, the medium comprises, consists of, orconsists essentially of the components of PAE-19 or PAE-21 listed inTable 12. In some embodiments, the medium comprises, consists of, orconsists essentially of the components of PAE-21. In some embodiments,the medium comprises, consists of, or consists essentially of thecomponents of PAE-19.

In various embodiments, the date palm explant tissue in the methodsdescribed above is from a male Black Barhee date palm. In someembodiments, the Black Barhee date palm explant tissue in the methodsdescribed above is from a female Black Barhee date palm.

In various embodiments, the Black Barhee date palm explant in themethods described above includes plant tissue from an offshoot of aBlack Barhee date palm. In some embodiments, the Black Barhee date palmexplant includes plant tissue from a flower, a leaf, or a bud of a BlackBarhee date palm. In some embodiments, the explant tissue, whether froman offshoot, a flower, a leaf, or a bud, comprises meristematic cells.

In various embodiments, the invention teaches a method formicropropagating a Black Barhee date palm. In some embodiments, themethod includes initiating a shoot in vitro from a Black Barhee datepalm explant on a first medium, and multiplying (i.e. growing, becausenow an additional shoot is being produced) the shoot initiated from theexplant in vitro on a second medium. In some embodiments, the firstmedium comprises, consists of, or consists essentially of theconstituents of M7, 1/2M7, M20, 1/2M20, M26, M27, M28, M29, M52, 1/2M52,M53, M63, PAE-1, PAE1-M, PAE-9, PAE-7, PAE-71, or any medium of Tables7-13. In some embodiments, the second medium comprises, consists of, orconsists essentially of the constituents of M7, M36, PAE-21, PAE-19, orany medium of Tables 7-13, or any medium of Tables 7-13 in which theconcentration of hormones has been reduced to ¾ to ⅛ of the initialconcentration listed in the table, or ½ to ⅛ of the initialconcentration listed in the table. In some embodiments, theconcentrations of hormones in any medium listed in Table 5 or Tables7-13 are reduced to ⅕ of the initial concentration listed in the tableduring the multiplication stage. In some embodiments, the second mediumcomprises, consists of, or consists essentially of the constituents ofM7, M36, any medium of Tables 7-13, or any medium of Tables 7-13 inwhich the concentrations of all constituents except the organics hasbeen reduced to ¾ to ⅛ of the initial concentration listed in the table,or ½ to ⅛ of the initial concentration listed in the table. In someembodiments, the concentrations of all constituents except the organicsin any medium listed in Table 5 or Tables 7-13 are reduced to ⅕ of theinitial concentration listed in the table for the multiplication stage.In certain embodiments, the date palm explant includes plant tissue froman offshoot of a date palm. In certain embodiments the date palm explantincludes plant tissue from a flower of a date palm. In some embodiments,the plant tissue is obtained by any means described herein. In certainembodiments, the plant tissue is treated with an antioxidant solutionand/or sterilized by any method described herein.

In some embodiments, the method further includes transferring one ormore of the multiplied shoots (i.e. a grown shoot that is large enoughto be isolated and established on a rooting medium described herein) toa rooting medium that comprises, consists of, or consists essentially ofthe constituents of a medium of Table 6. In some embodiments, therooting medium is any rooting medium described herein.

In certain embodiments, the invention teaches a method formicropropagating a Black Barhee date palm by utilizing one or more mediacomprising, consisting of, or consisting essentially of the constituentsof a medium of Tables 4 or 7-13 during the initiation stage (asdescribed herein). In some embodiments, the invention teaches a methodfor micropropagating a Black Barhee date palm by utilizing one or moremedia comprising, consisting of, or consisting essentially of theconstituents of a medium of Table 5, Tables 7-13, or modified versionsof any medium of Tables 7-9 in which the concentrations of one or morehormones and/or other constituents have been reduced as described abovefor the multiplication stage (as described herein). In certainembodiments, the invention teaches a method for micropropagating a BlackBarhee date palm by utilizing one or more media comprising, consistingof or consisting essentially of the constituents of a medium of Table 6for the rooting stage (as described herein). In some embodiments one ormore media utilized for the rooting stage comprises, consists of, orconsists essentially of the constituents of any medium described hereinas a rooting medium. In some embodiments, the invention teachesutilizing one or more media comprising, consisting of or consistingessentially of the constituents of one or more media of Tables 4 or 7-13for the initiation stage and/or one or more media comprising, consistingof, or consisting essentially of the constituents of one or more mediaof Table 5, Table 7-13, or modified versions of any medium of Tables7-13 in which the concentrations of hormones and/or other constituentshave been reduced as described above for the multiplication stage and/orone or more medium comprising, consisting of, or consisting essentiallyof the constituents of one or more media of Table 6 or any other mediumdescribed herein for the rooting stage.

In some embodiments, the invention teaches a kit that includes one ormore media comprising, consisting of, or consisting essentially of theconstituents of one or more media described in any of Tables 4-13. Insome embodiments, the kit includes one or more media comprising,consisting of, or consisting essentially of the constituents of one ormore media described in Tables 4-13 and/or one or more media comprising,consisting of, or consisting essentially of the constituents of one ormore media described in Table 5 or a low (reduced) hormone and/or otherconstituents version of any medium described in tables 4-13 (asdescribed above) and/or one or more media comprising, consisting of, orconsisting essentially of the constituents of one or more mediadescribed in Table 6 or any rooting medium described herein.

In some embodiments, the kit further includes a tissue sample from anysource described herein, obtained by any method described herein, andoptionally treated with any antioxidant solution described herein and/orsterilized by any method described herein. In some embodiments thetissue sample includes meristematic cells of a Black Barhee date palm.

In certain embodiments, the kit includes one or more containersconfigured for performing one or more stages of the tissue culturingprocess described herein (i.e. initiation, multiplication, or rooting).Non-limiting examples of tissue culture dishes are depicted in thedrawings of the present application.

In some embodiments, the kit includes one or more containers for sterilestorage of one or more media and/tissue sample and/or explant describedherein. In certain embodiments, the different components of the one ormore media can be packaged in separate containers and mixed before use.In certain embodiments, one or more media can be provided pre-mixed andoptionally marked with an expiration date for convenient storage anduse.

In some embodiments, the kit further includes instructional materialsfor utilizing all or a portion of the contents contained therein. Insome embodiments, the instructional materials may be printed on paper orother substrate, and/or may be supplied as an electronic-readable media,such as a floppy disc, CD-ROM, DVD-ROM, Zip disc, videotape, audiotape,etc. Detailed instructions may not be physically associated with thekit; instead, a user may be directed to an internet web site specifiedby the manufacturer or distributor of the kit, or supplied by electronicmail.

In some embodiments, the concentrations of one or more constituents ofthe media described herein above and below, and utilized in conjunctionwith the media/compositions, methods and kits described herein areconsistent with those listed in the referenced tables. In otherembodiments, the concentrations of one or more constituents of the mediadescribed herein above and below and utilized in conjunction with theinventive media/compositions, methods and kits are up to approximately+20% of the values stated in the tables.

In some embodiments, the invention teaches a Black Barhee date palmplant, a Black Barhee date palm tissue, or a Black Barhee date palm cellresulting from the process of micropropagation, or a stage within theprocess of micropropagation, according to one or more method describedherein and/or by utilizing one or more medium described herein duringone or more stage of micropropagation. The inventive plant, planttissue, or plant cell is intended to include a plant, plant tissue orplant cell at any stage of development after any stage of the process ofmicropropagation described herein has started. Thus, for example, aplant, a plant tissue, or a plant cell that results from initiation,multiplication, rooting, acclimation, or that has been temporarily orpermanently planted in soil, after one or more of the protocolsdescribed herein (or one or more portions thereof) has been applied, iswithin the scope of the present invention. Furthermore, a plant, planttissue, or plant cell that results after exposing starting material of aBlack Barhee date palm to any one or more medium that includes theconstituents of any one or more medium described herein, is within thescope of the present invention. In some embodiments, the startingmaterial includes all or a portion of a flower of a Black Barhee datepalm. In some embodiments, the starting material includes all or aportion of a bud of a Black Barhee date palm. In some embodiments, thestarting material includes a portion of an offshoot of a Black Barheedate palm. In some embodiments, the starting material includes a cell ofa Black Barhee date palm.

In some embodiments, the invention teaches a Black Barhee date palmplant resulting after exposing a tissue or a cell of a Black Barhee datepalm to media comprising, consisting of, or consisting essentially ofthe constituents of a medium selected from PAE-1, PAE-1M, PAE-9, PAE-71,PAE-19, PAE-21 and PAE-7. In some embodiments, one or more of theconstituents of the aforementioned media to which the tissue or a cellof a Black Barhee date palm has been exposed is varied by +20%. Incertain embodiments, the origin of the tissue is an offshoot, a flower,a leaf, or a bud.

In some embodiments, the invention teaches a Black Barhee date palmplant cell resulting from exposing a tissue or a cell of a Black Barheedate palm (e.g. as described herein) to media comprising, consisting of,or consisting essentially of the constituents of a medium selected fromthe group consisting of PAE-1, PAE-1M, PAE-9, PAE-21, PAE-71 and PAE-7.In some embodiments, one or more of the constituents of theaforementioned media to which the tissue or a cell of a Black Barheedate palm has been exposed is varied by +20%.

In some embodiments, the inventive tissue or cell originates from acallus formed on an explant of a Black Barhee date palm treated with oneor more media types described herein. In some embodiments, the explantincludes tissue from a flower, a bud, a leaf, or an offshoot of a BlackBarhee date palm.

In various embodiments, the invention teaches a Black Barhee date palmplant resulting from exposing a globule of a callus of a Black Barheedate palm explant to a medium comprising, consisting of, or consistingessentially of the constituents of a medium selected from PAE-1, PAE-1M,PAE-9, PAE-19, PAE-21, PAE-71 and PAE-7, wherein a starting materialfrom which the callus is formed is a flower, a bud, a leaf, or anoffshoot of a Black Barhee date palm. In some embodiments, one or moreof the constituents of the aforementioned media to which the globule ofa callus of the Black Barhee date palm explant has been exposed isvaried by +20%.

In some embodiments, the present invention teaches a method forgenerating revenue for a person or an entity (corporation, company, andthe like) by selling one or more micropropagated plant, a plant tissue,or a plant cell, at any stage described herein, resulting from any oneor more method described herein and/or by using any one or more mediumdescribed herein. In some embodiments, the invention teaches selling aplant that has been micropropagated according to one or more methodsdescribed herein. In some embodiments, the method teaches selling aportion of a plant, a cell of a plant, or group of cells of a plant. Insome embodiments, the plant, plant cell, or plant tissue is of BlackBarhee date palm that has been micropropagated according to any methoddescribed herein, or that is at any stage of the micropropagationprocess described herein, and/or that has been treated with (i.e.exposed to) any of one or more medium described herein.

EXAMPLES Example 1 Micropropagation of a Black Barhee Date PalmOrganogenesis

Selected offshoots of a Black Barhee date palm are 3-6 years old with anaverage weight of 3-10 kg. Before removing the offshoots, the connectionbetween the offshoots and mother plant is established. Shoot tips andbuds are obtained as described above. Alternatively, flowers may beprepared as described above.

Antioxidant Treatment

Shoot tips or flower tissue is transferred to an antioxidant solutioncontaining 100 mg of ascorbic acid and 150 mg of citric acid to avoidphenolic oxidation.

Incubation

Disinfected shoot tips and buds or flowers are transferred to anincubation media selected from one or more of PA01, PA02, PA03, PA04,PA05, PA06, PA07, PAE-1, PAE-1M, PAE-7, PAE-71, and PAE-9, as describedin Table 9. These explants are then incubated for 4-6 months, andsubcultured in fresh media approximately every month. After 4-6 monthsof incubation, the transplants are transferred to light with aphotoperiod of 16 h at 25° C. Optionally, PAE-9 can be used for theinitiation stage of an explant derived from flower starting material,whereas PAE-1M can be used for the initiation stage of an explantderived from shoot starting material (“shoot tip” starting material).

Shoot Multiplication

For the multiplication stage, ⅕ concentration of all constituents(except Nicotinic acid, Thiamine HCl, Pyridoxine HCl, Glycine,Glutamine, Adenine, Ca-pantothenate, and myoinositol) of any of PA01,PA02, PA03, PA04, PA05, PA06 and PA07 can be used (applied to initiatedmaterial). Alternatively, for multiplication, PAE-21 or PAE-19 can beused (applied to initiated material).

Shoot Elongation

Multiplied shoots are transferred to a MS media containing NAA 1 mg/L,BA 0.5 mg/L, Kinetin 0.5 mg/L and IBA 0.5 mg/L.

Rooting

Elongated shoots are rooted in a medium containing NAA 0.1 mg/L in thepresence of 40-60 g/L of sucrose.

Media Tables

TABLE 1 Major Salts (Macronutrients) Component mg/L Ammonium nitrate(NH₄NO₃) 1,650 ± 2   Calcium chloride (CaCl₂ 2H₂O) 440 ± 2 Magnesiumsulphate (MgSO₄ 7H₂O) 370 ± 2 Potassium phosphate (KH₂PO₄) 170 ± 2Potassium nitrate (KNO₃) 1,900 ± 2  

TABLE 2 Minor Salts (Micronutrients) Component mg/L Boric acid (H₃BO₃) 6.2 ± 0.2 Cobalt chloride (CoCl₂ 6H₂O) 0.025 ± .002 Cupric sulphate(CuSO₄ 5H₂O) 0.025 ± .002 Ferrous sulphate (FeSO₄ 7H₂O) 27.8 ± 0.2Manganese sulphate (MnSO₄ 4H₂O) 22.3 ± 2   Potassium iodide (KI) 0.83 ±.02 Sodium molybdate (Na₂MoO₄ 2H₂O) 0.25 ± .02 Zinc sulphate(ZnSO₄•7H₂O)  8.6 ± 0.2 Ethylenediaminetetraacetic acid (Na₂EDTA 2H₂O)37.2 ± 2  

TABLE 3 Organics Component mg/L (unless otherwise noted) Myo-Inositol100 ± 2  Thiamine 0.4 ± 0.3 Pyridoxine 0.5 ± 0.2 Nicotinic acid 0.5 ±0.2 Glycine   2 ± 0.5 Sucrose 30 g/L ± 2

TABLE 4 Initiation Media I Modification to MS Salts (If not modifiedfrom concentrations Medium in Tables Modification(s) to Organics(Hormones, Name 1 & 2 “NA”) Vitamins, Sugars, Agar, etc.) M7 NA  1. BAP= 1 ± 0.2 mg/L  2. Charcoal = 500 ± 50 mg/L 1/2M7 50% MS Salts  1. BAP =1 ± 0.2 mg/L  2. Charcoal = 500 ± 50 mg/L M20 NA  1. BAP = 3 ± 0.2 mg/L 2. NAA = 1 ± 0.2 mg/L  3. Charcoal = 500 ± 50 mg/L 1/2M20 50% MS Salts 1. BAP = 3 ± 0.2 mg/L  2. NAA = 1 ± 0.2 mg/L  3. Charcoal = 500 ± 50mg/L M26 NA  1. NaH₂PO₄ = 170 ± 10 mg/L  2. Glutamine = 200 ± 10 mg/L 3. BAP = 1 ± 0.2 mg/L  4. 2iP = 2 ± 0.2 mg/L  5. NAA = 1 ± 0.2 mg/L  6.NOA = 1 ± 0.2 mg/L  7. Ascorbic acid = 50 ± 5 mg/L  8. Charcoal = 500 ±50 mg/L M27 NA  1. NaH₂PO₄ = 170 ± 10 mg/L  2. Glutamine = 200 ± 10 mg/L 3. BAP = 5 ± 0.2 mg/L  4. 2iP = 1 ± 0.2 mg/L  5. NAA = 0.1 mg/L  6. NOA= 0.5 ± 0.2 mg/L  7. Ascorbic acid = 50 ± 5 mg/L  8. Charcoal = 500 ± 50mg/L M28 NA  1. NaH₂PO₄ = 170 ± 10 mg/L  2. Glutamine = 200 ± 10 mg/L 3. BAP = 1 ± 0.2 mg/L  4. 2iP = 1 ± 0.2 mg/L  5. Kinetin = 1 ± 0.2 mg/L 6. NOA = 0.5 ± 0.2 mg/L  7. Ascorbic acid = 50 ± 5 mg/L  8. Charcoal =500 ± 50 mg/L M29 NA  1. NaH₂PO₄ = 170 ± 10 mg/L  2. Glutamine = 200 ±10 mg/L  3. BAP = 1 ± 0.2 mg/L  4. 2iP = 2 ± 0.2 mg/L  5. NOA = 0.5 ±0.2 mg/L  6. Ascorbic acid = 50 ± 5 mg/L  7. Charcoal = 500 ± 50 mg/LM52 NA  1. NaH₂PO₄ = 170 ± 10 mg/L  2. Thiamine HCl = 5 ± 1 mg/L  3.Nicotinic acid = 5 ± 1 mg/L  4. Pyridoxine HCl = 2.5 ± .5 mg/L  5.Ca-pantothenate = 2.5 ± .5 mg/L  6. Inositol = 10 ± 1 mg/L  7. Glycine =3 ± .2 mg/L  8. Glutamine = 20 ± 2 mg/L  9. NAA = 5 ± 1 mg/L 10. NOA = 5± 1 mg/L 11. BAP = 1 ± .1 mg/L 12. Kinetin = 1 ± .1 mg/L 13. 2iP = 1 ±.1 mg/L 14. Citric acid = 75 ± 5 mg/L 15. Ascorbic acid = 75 ± 5 mg/L16. Charcoal = 1.5 g/L 17. Agar (Gelrite) = 1.3 g/L 18. pH = 5.6 ± .11/2M52 50% MS salts  1. NaH₂PO₄ = 170 ± 10 mg/L  2. Thiamine HCl = 5 ± 1mg/L  3. Nicotinic acid = 5 ± 1 mg/L  4. Pyridoxine HCl = 2.5 ± .5 mg/L 5. Ca-pantothenate = 2.5 ± .5 mg/L  6. Inositol = 10 ± 1 mg/L  7.Glycine = 3 ± .2 mg/L  8. Glutamine = 20 ± 2 mg/L  9. NAA = 5 ± 1 mg/L10. NOA = 5 ± 1 mg/L 11. BAP = 1 ± .1 mg/L 12. Kinetin = 1 ± .1 mg/L 13.2iP = 1 ± .1 mg/L 14. Citric acid = 75 ± 5 mg/L 15. Ascorbic acid = 75 ±5 mg/L 16. Charcoal = 1.5 g/L 17. Agar (Gelrite) = 1.3 g/L, pH = 5.6 ±.1 M53 NA  1. NaH₂PO₄ = 10 ± 1 mg/L  2. Thiamine HCl = 5 ± 1 mg/L  3.Nicotinic acid = 5 ± 1 mg/L  4. Pyridoxine HCl = 2.5 ± .5 mg/L  5.Ca-pantothenate = 2.5 ± .5 mg/L  6. Inositol = 10 ± 1 mg/L  7. Glycine =3 ± .2 mg/L  8. Glutamine = 20 ± 2 mg/L  9. 2,4-D = 1 ± .1 mg/L 10.Kinetin = 3 ± .1 mg/L 11. 2iP = 3 ± .1 mg/L 12. Citric Acid = 75 ± 5mg/L 13. Ascorbic Acid = 75 ± 5 mg/L 14. Charcoal = 500 ± 50 mg/L M63 NA 1. Nicotinic Acid = 0.5 ± .1 mg/L  2. Pyridoxine HCl = 0.5 ± .1 mg/L 3. Thiamine = 0.5 ± .1 mg/L  4. Glycine = 2 ± .1 mg/L  5. Inositol = 10± 1 mg/L  6. Glutamine = 10 ± 1 mg/L  7. Adenine = 30 ± 2 mg/L  8. 2,4-D= 1 ± .1 mg/L  9. Charcoal = 500 ± 50 mg/L

TABLE 5 Multiplication Media I Modification(s) to MS Salts (if notmodified from Modification(s) to Medium concentrations in Tables 1&2Organics (Hormones, Name “NA”) Vitamins, Sugars, Agar, etc.) M7  NA 1.BAP = 1 ± 0.2 mg/L 2. Charcoal = 500 ± 50 mg/L M36 NA 1. NaH₂PO₄ = 150 ±10 mg/L 2. Glutamine = 200 ± 10 mg/L 3. BAP = 2 ± 0.2 mg/L 4. 2iP = 4 ±0.2 mg/L 5. NAA = 1 ± 0.2 mg/L 6. NOA = 1 ± 0.2 mg/L 7. Charcoal = 500 ±50 mg/L

TABLE 6 Rooting Media Modification(s) to MS Salts (if not modified fromModification(s) to Medium concentrations in Tables 1&2 Organics(Hormones, Name “NA”) Vitamins, Sugars, Agar, etc.) M70 NA 1. NAA = 1 ±0.2 mg/L 2. Agar (Gelrite) = 1.5 ± 0.2 g/L M82 NA 1. NAA = 0.1 ± .02mg/L 2. Charcoal = 500 ± 50 mg/L

For each type of medium listed in Tables 7-9, the amount of eachcomponent may be adjusted by up to +20%, with the volume of wateradjusted appropriately such that the total volume of for each mediumprepared is 1 L. One of skill in the art would understand that therecipe for each medium type can be adjusted appropriately if a totalvolume of less or more than 1 L is desired for any particularapplication. For each table herein, “BTV 1 L” means that a quantity ofwater is added to the medium recipe such that the total volume of themedium is 1 L.

For each medium listed in Tables 7-9 each of the major salts of Table 1and minor salts of Table 2 are added according to the concentrationslisted in Table 1 and Table 2. In addition, for each medium listed inTables 7-9 each of the organics listed in Table 3 are added according tothe concentrations listed in Table 3. Any additional major salts, minorsalts, and organics that are included in the media of Tables 7-9 arespecifically listed in those tables with appropriate concentrations orranges of concentrations noted.

TABLE 7 Initiation Media II Medium Type PAEX1 PAEX2 PAEX3 PAEX4 PAEX5PAEX6 PAEX7 PAEX8 DI Water (ml/L) BTV 1 L BTV 1 L BTV 1 L BTV 1 L BTV 1L BTV 1 L BTV 1 L BTV 1 L Glutamine (mg/L) 10 10 10 10 10 10 10 10Ca-Pantothenate (mg/L) 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 Adenine (mg/L) 33 3 3 3 3 3 3 Ascorbic Acid (mg/L) 75 75 75 75 75 75 75 75 BAP (mg/L) 15 3 5 IAA (mg/L) Kinetin (mg/L) NOA (mg/L) NAA (mg/L) 2,4 D (mg/L) 1 52iP (mg/L) 3 3 3 Activated charcoal (g/L) 0.5 0.5 0.5 0.5 0.5 0.5 0.50.5 Agar (g/L) 6.6 6.6 6.6 6.6 6.6 6.6 6.6 6.6

TABLE 8 Initiation Media III Medium Type PAEX9 PAEX10 PAEX11 PAEX12PAEX13 PAEX14 PAEX15 DI Water (ml/L) BTV 1 L BTV 1 L BTV 1 L BTV 1 L BTV1 L BTV 1 L BTV 1 L Glutamine (mg/L) 10 10 10 10 10 10 10Ca-Pantothenate (mg/L) 2.5 2.5 2.5 2.5 2.5 2.5 2.5 Adenine (mg/L) 3 3 33 3 3 3 Ascorbic Acid (mg/L) 75 75 75 75 75 75 75 BAP (mg/L) 5 5 5 5 IAA(mg/L) 1 1 Kinetin (mg/L) 3 3 3 3 NOA (mg/L) 5 5 5 NAA (mg/L) 1 1 2,4 D(mg/L) 1 5 2iP (mg/L) 3 3 3 3 Activated charcoal (g/L) 0.5 0.5 0.5 0.50.5 0.5 0.5 Agar (g/L) 6.6 6.6 6.6 6.6 6.6 6.6 6.6

TABLE 9 Initiation Media IV Medium Type PA01 PA02 PA03 PA04 PA05 PA06PA07 DI Water (ml/L) BTV 1 L BTV 1 L BTV 1 L BTV 1 L BTV 1 L BTV 1 LBTV1 L Glutamine (mg/L) 10 10 10 10 10 10 10 Ca-Pantothenate (mg/L) 2.52.5 2.5 2.5 2.5 2.5 2.5 Adenine (mg/L) 3 3 3 3 3 3 3 Ascorbic Acid(mg/L) 75 75 75 75 75 75 75 IBA (mg/L) 0.2 BAP (mg/L) 1 1 IAA (mg/L) 0.51 1 Kinetin (mg/L) 1 NOA (mg/L) 1 1 3 NAA (mg/L) 0.5 1 1 0.1 1 0.5 2,4 D(mg/L) 2iP (mg/L) 0.2 0.2 2 2 0.5 1 Activated charcoal (g/L) 0.5 0.5 0.50.5 0.5 0.5 0.5 Agar (g/L) 6.6 6.6 6.6 6.6 6.6 6.6 6.6

TABLE 10 Initiation Media V Media - Components Unit PAE-1 PAE-9 PAE-7Distilled Water mL 990.0 990.0 990.0 Sucrose g/L 30.0 30.0 30.0 AmmoniumNitrate (NH₄NO₃) mg/L 825.0 825.0 825.0 Boric Acid (H₃BO₃) mg/L 3.103.10 3.10 Calcium Chloride, Anhydrous (CaCl₂) mg/L 166.050 166.050166.050 Cobalt Chloride, Hexahydrate (CoCl₂•6H₂0) mg/L 0.0125 0.01250.0125 Cupric Sulfate, Pentahydrate (CuSO₄•5H₂O) mg/L 0.0125 0.01250.0125 EDTA, Disodium, Dihydrate (C₁₀H₁₄N₂Na₂O₈•2H₂0) mg/L 18.630 18.63018.630 Ferrous Sulfate, Heptahydrate (FeSO₄•7H₂O) mg/L 13.90 13.90 13.90Magnesium Sulfate, Anhydrous (MgSO₄) mg/L 90.350 90.350 90.350 MagnesiumSulfate, Monohydrate (MgSO₄•H₂O) mg/L 8.450 8.450 8.450 Molybdic AcidSodium Salt, Dihydrate (NaMoO₄•2H₂O) mg/L 0.1250 0.1250 0.1250 PotassiumIodide (KI) mg/L 0.4150 0.4150 0.4150 Potassium Nitrate (KNO₃) mg/L950.0 950.0 950.0 Potassium Phosphate, Monobasic, Anhydrous (KH₂PO₄)mg/L 85.0 85.0 85.0 Zinc Sulfate, Heptahydrate (ZnSO₄•7H₂O) mg/L 4.304.30 4.30 Myo-Inositol (C₆H₅NO₂) mg/L 100.0 100.0 100.0 Glycine(C₂H₅NO₂) mg/L 2.0 2.0 2.0 Nicotinic Acid (C₆H₅NO₂) mg/L 0.50 0.50 0.50Pyridoxine, Hydrochloride (C₈H₁₁NO₃•HCL) mg/L 0.50 0.50 0.50 Thiamine,Hydrochloride (C₁₂H₁₇CIN₄OS•HCL) mg/L 0.10 0.10 0.10 Citric Acid mg/L75.0 75.0 75.0 Ascorbic Acid mg/L 75.0 75.0 75.0 Glutamine mg/L 200.0200.0 200.0 2,4-Dichlorophenoxyacetic Acid (2,4-D) mg/L — 1.0 —N6-(2-Isopentenyl)adenine (2-iP) mg/L 2.0 3.0 — 6-Benzylaminopurine(BAP) mg/L 1.0 0.1 Kinetin (KIN) mg/L — 3.0 0.01 3-Indoleacetic Acid(IAA) mg/L — — — Naphthaleneacetic Acid (NAA) mg/L — — 1.02-Naphthoxyacetic acid (NOA) mg/L 5.0 — Activated Charcoal g/L 0.5 0.50.5 Agar, Powder (e.g., Powder Micropropagation Type II - g/L 7.0 7.07.0 Caisson) pH — 5.7 5.7 5.7

TABLE 11 Initiation Media VI Media - Components Unit M26 M28 M40 M46Distilled Water mL 990.0 990.0 990.0 990.00 Sucrose g/L 30.0 30.0 30.030.00 Ammonium Nitrate (NH₄NO₃) mg/L 825.0 1650.0 1650.0 1650.0 BoricAcid (H₃BO₃) mg/L 3.10 6.20 6.20 6.20 Calcium Chloride, Anhydrous(CaCl₂) mg/L 166.050 322.10 322.10 322.10 Cobalt Chloride, Hexahydrate(CoCl₂•6H₂0) mg/L 0.0125 0.0250 0.0250 0.0250 Cupric Sulfate,Pentahydrate (CuSO₄•5H₂O) mg/L 0.0125 0.0250 0.0250 0.0250 EDTA,Disodium, Dihydrate (C₁₀H₁₄N₂Na₂O₈•2H₂0) mg/L 18.630 37.260 37.26037.260 Ferrous Sulfate, Heptahydrate (FeSO₄•7H₂O) mg/L 13.90 27.80 27.8027.80 Magnesium Sulfate, Anhydrous (MgSO₄) mg/L 90.350 180.70 180.70180.70 Magnesium Sulfate, Monohydrate (MgSO₄•H₂O) mg/L 8.450 16.90 16.9016.90 Molybdic Acid Sodium Salt, Dihydrate (NaMoO₄•2H₂O) mg/L 0.12500.250 0.250 0.250 Potassium Iodide (KI) mg/L 0.4150 0.4150 0.4150 0.4150Potassium Nitrate (KNO₃) mg/L 950.0 1900.0 1900.0 1900.0 PotassiumPhosphate, Monobasic, Anhydrous (KH₂PO₄) mg/L 85.0 170.0 170.0 170.0Zinc Sulfate, Heptahydrate (ZnSO₄•7H₂O) mg/L 4.30 8.60 8.60 8.60Myo-Inositol (C₆H₅NO₂) mg/L 100.0 100.0 100.0 100.0 Glycine (C₂H₅NO₂)mg/L 2.0 2.0 2.0 2.0 Nicotinic Acid (C₆H₅NO₂) mg/L 0.50 0.50 0.50 0.50Pyridoxine, Hydrochloride (C₈H₁₁NO₃•HCL) mg/L 0.50 0.50 0.50 0.50Thiamine, Hydrochloride (C₁₂H₁₇CIN₄OS•HCL) mg/L 0.10 0.10 0.10 0.10Citric Acid mg/L 0.00 0.00 0.00 0.00 Ascorbic Acid mg/L 50.0 50.00 0.000.00 Glutamine mg/L 200.0 200.0 200.0 200.0 2,4-DichlorophenoxyaceticAcid (2,4-D) mg/L — 0.1 5.0 N6-(2-Isopentenyl)adenine (2-iP) mg/L — 1.00.5 6-Benzylaminopurine (BAP) mg/L 1.0 1.0 — — Kinetin (KIN) mg/L 1.03-Indoleacetic Acid (IAA) mg/L — — 0.1 Naphthaleneacetic Acid (NAA) mg/L1.0 — 1.0 5.0 2- Naphthoxyacetic acid (NOA) mg/L 1.0 0.5 — ActivatedCharcoal g/L 0.5 Agar, Powder (e.g., Micropropagation Type II - Caisson)g/L 7.0 7.0 7.0 7.0 pH — 5.7 5.7 5.7 5.7

TABLE 12 Multiplication Media II Media - Components Unit PAE-13 PAE-15PAE-17 PAE-19 PAE-21 Distilled Water mL 990.0 990.0 990.0 990.0 990.0Sucrose g/L 30.0 30.0 30.0 30.0 30.0 Ammonium Nitrate (NH₄NO₃) mg/L825.0 825.0 825.0 1650.0 1650.0 Boric Acid (H₃BO₃) mg/L 3.10 3.10 3.106.20 6.20 Calcium Chloride, Anhydrous mg/L 166.050 166.050 166.050322.10 322.10 (CaCl₂) Cobalt Chloride, Hexahydrate mg/L 0.0125 0.01250.0125 0.0250 0.0250 (CoCl₂•6H₂0) Cupric Sulfate, Pentahydrate mg/L0.0125 0.0125 0.0125 0.0250 0.0250 (CuSO₄•5H₂O) EDTA, Disodium,Dihydrate mg/L 18.630 18.630 18.630 37.260 37.260 (C₁₀H₁₄N₂Na₂O₈•2H₂0)Ferrous Sulfate, Heptahydrate mg/L 13.90 13.90 13.90 27.80 27.80(FeSO₄•7H₂O) Magnesium Sulfate, Anhydrous mg/L 90.350 90.350 90.350180.70 180.70 (MgSO₄) Magnesium Sulfate, Monohydrate mg/L 8.450 8.4508.450 16.90 16.90 (MgSO₄•H₂O) Molybdic Acid Sodium Salt, mg/L 0.12500.1250 0.1250 0.250 0.250 Dihydrate (NaMoO₄•2H₂O) Potassium Iodide (KI)mg/L 0.4150 0.4150 0.4150 0.830 0.4150 Potassium Nitrate (KNO₃) mg/L950.0 950.0 950.0 1900.0 1900.0 Potassium Phosphate, Monobasic, mg/L85.0 85.0 85.0 170.0 170.0 Anhydrous (KH₂PO₄) Zinc Sulfate, Heptahydratemg/L 4.30 4.30 4.30 8.60 8.60 (ZnSO₄•7H₂O) Myo-Inositol (C₆H₅NO₂) mg/L100.0 100.0 100.0 100.0 100.0 Glycine (C₂H₅NO₂) mg/L 2.0 2.0 2.0 2.0 2.0Nicotinic Acid (C₆H₅NO₂) mg/L 0.50 0.50 0.50 0.50 0.50 Pyridoxine,Hydrochloride mg/L 0.50 0.50 0.50 0.50 0.50 (C₈H₁₁NO₃•HCL) Thiamine,Hydrochloride mg/L 0.10 0.10 0.10 0.10 0.10 (C₁₂H₁₇CIN₄OS•HCL) CitricAcid mg/L 75.0 75.0 75.0 0.0 0.0 Ascorbic Acid mg/L 75.0 75.0 75.0 0.00.0 Glutamine mg/L 200.0 200.0 200.0 200.0 200.02,4-Dichlorophenoxyacetic Acid mg/L — — — (2,4-D)N6-(2-Isopentenyl)adenine (2-iP) mg/L 1.0 — 6-Benzylaminopurine (BAP)mg/L 5.0 1.0 3.0 1.0 1.0 Kinetin (KIN) mg/L — 1.0 3-Indoleacetic Acid(IAA) mg/L 1.0 — — Naphthaleneacetic Acid (NAA) mg/L 0.1 — 1.0 —2-Naphthoxyacetic acid (NOA) mg/L 0.5 0.5 — Activated Charcoal g/L 0.50.5 0.5 0.5 Agar, Powder (e.g., Powder g/L 7.0 7.0 7.0 7.0 7.0Micropropagation Type II - Caisson) pH — 5.7 5.7 5.7 5.7 5.7

TABLE 13 Multiplication Media III Media - Components Unit M21 M36Distilled Water mL 990.0 990.0 Sucrose g/L 30.0 30.0 Ammonium Nitrate(NH₄NO₃) mg/L 1650.0 1650.0 Boric Acid (H₃BO₃) mg/L 6.20 6.20 CalciumChloride, Anhydrous (CaCl₂) mg/L 322.10 322.10 Cobalt Chloride,Hexahydrate (CoCl₂•6H₂0) mg/L 0.0250 0.0250 Cupric Sulfate, Pentahydrate(CuSO₄•5H₂O) mg/L 0.0250 0.0250 EDTA, Disodium, Dihydrate(C₁₀H₁₄N₂Na₂O₈•2H₂0) mg/L 37.260 37.260 Ferrous Sulfate, Heptahydrate(FeSO₄•7H₂O) mg/L 27.80 27.80 Magnesium Sulfate, Anhydrous (MgSO₄) mg/L180.70 180.70 Magnesium Sulfate, Monohydrate (MgSO₄•H₂O) mg/L 16.9016.90 Molybdic Acid Sodium Salt, Dihydrate (NaMoO₄•2H₂O) mg/L 0.2500.250 Potassium Iodide (KI) mg/L 0.4150 0.8300 Potassium Nitrate (KNO₃)mg/L 1900.0 1900.0 Potassium Phosphate, Monobasic, Anhydrous (KH₂PO₄)mg/L 170.0 170.0 Zinc Sulfate, Heptahydrate (ZnSO₄•7H₂O) mg/L 8.60 8.60Myo-Inositol (C₆H₅NO₂) mg/L 100.0 100.0 Glycine (C₂H₅NO₂) mg/L 2.0 2.0Nicotinic Acid (C₆H₅NO₂) mg/L 0.50 0.50 Pyridoxine, Hydrochloride(C₈H₁₁NO₃•HCL) mg/L 0.50 0.50 Thiamine, Hydrochloride (C₁₂H₁₇CIN₄OS•HCL)mg/L 0.10 0.10 Citric Acid mg/L 0.00 0.00 Ascorbic Acid mg/L 0.00 0.00Glutamine mg/L 200.0 0.00 2,4-Dichlorophenoxyacetic Acid (2,4-D) mg/L4.0 N6-(2-Isopentenyl)adenine (2-iP) mg/L 0.5 6-Benzylaminopurine (BAP)mg/L 3.0 2.0 Kinetin (KIN) mg/L 3-Indoleacetic Acid (IAA) mg/LNaphthaleneacetic Acid (NAA) mg/L 0.1 1.0 2-Naphthoxyacetic acid (NOA)mg/L — 1.0 Activated Charcoal g/L Agar, Powder (e.g., PowderMicropropagation Type II - Caisson) g/L 7.0 7.0 pH — 5.7 5.7

The various methods and techniques described above provide a number ofways to carry out the invention. Of course, it is to be understood thatnot necessarily all objectives or advantages described can be achievedin accordance with any particular embodiment described herein. Thus, forexample, those skilled in the art will recognize that the methods can beperformed in a manner that achieves or optimizes one advantage or groupof advantages as taught herein without necessarily achieving otherobjectives or advantages as taught or suggested herein. A variety ofalternatives are mentioned herein. It is to be understood that somepreferred embodiments specifically include one, another, or severalfeatures, while others specifically exclude one, another, or severalfeatures, while still others mitigate a particular feature by inclusionof one, another, or several advantageous features.

Furthermore, the skilled artisan will recognize the applicability ofvarious features from different embodiments. Similarly, the variouselements, features and steps discussed above, as well as other knownequivalents for each such element, feature or step, can be employed invarious combinations by one of ordinary skill in this art to performmethods in accordance with the principles described herein. Among thevarious elements, features, and steps some will be specifically includedand others specifically excluded in diverse embodiments.

Although the application has been disclosed in the context of certainembodiments and examples, it will be understood by those skilled in theart that the embodiments of the application extend beyond thespecifically disclosed embodiments to other alternative embodimentsand/or uses and modifications and equivalents thereof.

In some embodiments, the terms “a” and “an” and “the” and similarreferences used in the context of describing a particular embodiment ofthe application (especially in the context of certain of the followingclaims) can be construed to cover both the singular and the plural. Therecitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (for example, “such as”) provided withrespect to certain embodiments herein is intended merely to betterilluminate the application and does not pose a limitation on the scopeof the application otherwise claimed. No language in the specificationshould be construed as indicating any non-claimed element essential tothe practice of the application.

Preferred embodiments of this application are described herein,including the best mode known to the inventor for carrying out theapplication. Variations on those preferred embodiments will becomeapparent to those of ordinary skill in the art upon reading theforegoing description. It is contemplated that skilled artisans canemploy such variations as appropriate, and the application can bepracticed otherwise than specifically described herein. Accordingly,many embodiments of this application include all modifications andequivalents of the subject matter recited in the claims appended heretoas permitted by applicable law. Moreover, any combination of theabove-described elements in all possible variations thereof isencompassed by the application unless otherwise indicated herein orotherwise clearly contradicted by context.

All patents, patent applications, publications of patent applications,and other material, such as articles, books, specifications,publications, documents, things, and/or the like, referenced herein arehereby incorporated herein by this reference in their entirety for allpurposes, excepting any prosecution file history associated with same,any of same that is inconsistent with or in conflict with the presentdocument, or any of same that may have a limiting affect as to thebroadest scope of the claims now or later associated with the presentdocument. By way of example, should there be any inconsistency orconflict between the description, definition, and/or the use of a termassociated with any of the incorporated material and that associatedwith the present document, the description, definition, and/or the useof the term in the present document shall prevail.

In closing, it is to be understood that the embodiments of theapplication disclosed herein are illustrative of the principles of theembodiments of the application. Other modifications that can be employedcan be within the scope of the application. Thus, by way of example, butnot of limitation, alternative configurations of the embodiments of theapplication can be utilized in accordance with the teachings herein.Accordingly, embodiments of the present application are not limited tothat precisely as shown and described.

1. A method for micropropagating a Black Barhee date palm, comprising:applying a first medium in vitro to a Black Barhee date palm explanttissue comprising meristematic cells, at least until a shoot isinitiated, wherein the first medium comprises: (a)6-(gamma-gamma-dimethylallylamino)-purine (2-ip), 6-Benzylaminopurine(BAP) and Naphthoxyacetic acid (NOA); or (b) 2,4-Dichlorophenoxyaceticacid (2,4-D), 6-(gamma-gamma-dimethylallylamino)-purine (2-iP) andkinetic (KIN); or (c) 6-Benzylaminopurine (BAP), kinetic (KIN), and1-Naphthaleneacetic acid (NAA); and applying a second medium in vitro tothe initiated shoot, wherein the second medium comprises6-Benzylaminopurine (BAP).
 2. A method for micropropagating a BlackBarhee date palm, comprising: applying a first medium in vitro to aBlack Barhee date palm explant tissue comprising meristematic cells,until a shoot is initiated, wherein the first medium comprises: (a)6-(gamma-gamma-dimethylallylamino)-purine (2-ip), 6-Benzylaminopurine(BAP) and Naphthoxyacetic acid (NOA); or (b) 2,4-Dichlorophenoxyaceticacid (2,4-D), 6-(gamma-gamma-dimethylallylamino)-purine (2-iP) andkinetic (KIN); or (c) 6-Benzylaminopurine (BAP), kinetic (KIN), and1-Naphthaleneacetic acid (NAA).
 3. The method of claim 1, wherein thefirst and/or second media further comprises charcoal.
 4. The method ofclaim 1, wherein the first medium further comprises any of about 25%,about 50%, about 75%, about 90% or 100% of Murashige and Skoog standardconcentration of salts.
 5. A method for micropropagating a Black Barheedate palm, comprising applying a medium in vitro to an initiated BlackBarhee date palm shoot derived from an explant, wherein the mediumcomprises 6-Benzylaminopurine (BAP).
 6. The method of claim 5, whereinthe medium further comprises any of about 25%, about 50%, about 75%,about 90% or 100% of Murashige and Skoog standard concentration ofsalts.
 7. The method of claim 1, wherein the Black Barhee date palm is amale.
 8. The method of claim 1, wherein the Black Barhee date palm is afemale.
 9. The method of claim 1, wherein the Black Barhee date palmexplant comprises plant tissue from an offshoot of a Black Barhee datepalm.
 10. The method of claim 1, wherein the Black Barhee date palmexplant comprises plant tissue from a flower, a leaf, or a bud of aBlack Barhee date palm.
 11. A medium comprising the constituents ofPAE-1, PAE1-M, PAE-9, PAE-21, PAE-71, PAE-19 or PAE-7.
 12. A kit formicropropagating a Black Barhee date palm, the kit comprising one ormore media comprising the constituents of claim 11 selected from thegroup consisting of PAE-1, PAE-1M, PAE-71, PAE-9, PAE-19, PAE-21 andPAE-7.
 13. The kit of claim 12, further comprising a tissue of a BlackBarhee date palm.
 14. The kit of claim 13, wherein the tissue comprisesplant tissue from an offshoot, a flower, a leaf, or a bud of the BlackBarhee date palm.
 15. The kit of claim 12, further comprisinginstructions for the use of the one or more media for the purpose ofmicropropagating the Black Barhee date palm.
 16. A Black Barhee datepalm resulting from the method of claim
 1. 17. A Black Barhee date palmtissue resulting from the method of claim
 1. 18. A Black Barhee datepalm cell resulting from the method of claim
 1. 19. A Black Barhee datepalm plant resulting after exposing a tissue or a cell of a Black Barheedate palm to media comprising the constituents of claim 11 selected fromthe group consisting of PAE-1, PAE1-M, PAE-9, PAE-71, PAE-19, PAE-21 andPAE-7.
 20. The Black Barhee date palm plant of claim 19, wherein theorigin of the tissue is an offshoot, a flower, a leaf, or a bud.
 21. ABlack Barhee date palm tissue resulting after exposing a tissue or acell of a Black Barhee date palm to media comprising the constituents ofclaim 11 selected from the group consisting of PAE-1, PAE-1M, PAE-71,PAE-9, PAE-21, and PAE-7.
 22. The Black Barhee date palm plant tissue ofclaim 21, wherein the origin of the tissue of the Black Barhee date palmis an offshoot, a flower, a leaf, or a bud.
 23. A Black Barhee date palmplant cell resulting from exposing a tissue or a cell of a Black Barheedate palm to media comprising the constituents of claim 11 selected fromthe group consisting of PAE-1, PAE-1M, PAE-71, PAE-9, PAE-21, PAE-19 andPAE-7.
 24. The Black Barhee date palm plant cell of claim 23, whereinthe tissue or the cell originates from a callus formed on an explant ofa Black Barhee date palm.
 25. The Black Barhee date palm plant cell ofclaim 24, wherein the explant comprises tissue from a flower, a bud, aleaf, or an offshoot of a Black Barhee date palm.
 26. A Black Barheedate palm plant resulting from exposing a globule of a callus of a BlackBarhee date palm explant to a medium comprising the constituents ofclaim 11 selected from the group consisting of PAE-1, PAE-1M, PAE-9,PAE-19, PAE-21, and PAE-7, wherein a starting material from which thecallus is formed is a flower, a bud, a leaf, or an offshoot of a BlackBarhee date palm.